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Abstract

Introduction: The hypertrophic response to overload in fast-twitch skeletal muscle is impaired in aged humans and rats, and impaired protein synthesis pathway activation is hypothesized to be a contributing factor. Muscle growth occurs when protein synthesis exceeds protein degradation. Dietary supplementation of the essential amino acid leucine has been shown to enhance protein synthesis in both young and aged skeletal muscle. Leucine acts in part by activating mammalian target of rapamycin (mTOR; a key upstream regulator of protein synthesis pathways) as well as by attenuating the activation of 5'-AMP-activated protein kinase (AMPK; a negative regulator of mTOR and protein synthesis). During the aging process, AMPK Thr172 phosphorylation (and thus its activation) is increased, purportedly inhibiting gains in muscle mass and strength. Although dietary leucine supplementation has been shown to enhance strength gains in response to resistance training in young humans, the potential for leucine supplementation to enhance overload-induced muscle hypertrophy in aged humans or animal models has not been examined. Thus, the aim of this study was to determine whether dietary leucine supplementation can enhance markers of protein synthesis and rescue hypertrophy in overloaded fast-twitch skeletal muscles of aged rats to levels comparable to their younger counterparts. It was hypothesized that dietary leucine supplementation during 7 days of fast-twitch plantaris muscle overload would enhance plantaris muscle hypertrophy in aged rats to levels observed in young adult rats not receiving leucine. It was also hypothesized that dietary leucine supplementation during the overload period would suppress AMPK phosphorylation and enhance markers of protein synthesis [70 kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6), and eukaryotic elongation factor 2 (eEF2)] in the overloaded fast-twitch plantaris muscles of the aged rats to levels observed in young adult rats not receiving leucine.

Methods: Young adult (8 mo.) and old (33 mo.) male Fisher 344 x Brown Norway F1 Hybrid (FBN) rats underwent a 1-week unilateral overload of the fast-twitch plantaris muscles via tenotomy of the synergistic gastrocnemius muscle. Within each age group, animals were matched for body weight and separated into either a dietary leucine supplementation group (normal rat chow supplemented by an additional 5% leucine content in place of 5% of the carbohydrate content; n = 7/age group) or placebo group (normal rat chow; n = 6/age group). The leucine groups started the leucine-enriched diet 2 days prior to, and throughout, the overload intervention. All animals had ad libitum access to water and chow during the entire experiment; no differences in daily calorie consumption were observed between the placebo vs. leucine groups within each age group. At the end of the overload period, sham-operated and overloaded plantaris muscles were harvested and analyzed via western blotting for the phosphorylations of AMPK, p70S6k, rpS6, and eEF2. A 2x2x2 ANOVA with repeated measures was used for analyses of the effects of age, dietary intervention, and overload (the repeated measure) on muscle hypertrophy. A 2x2 ANOVA was used to measure the percent changes in hypertrophy and western blot analyses. Post-hoc comparisons were accomplished via a Fisher's Least Significant Difference test, with statistical significance being set at p ≤ 0.05.

Results: Dietary leucine enrichment significantly (p ≤ 0.05) enhanced overload-induced fast-twitch plantaris muscle hypertrophy in old, but not in young adult, animals. A similar effect was also observed in the slow-twitch soleus muscles, but western blotting analyses are only presented for the fast-twitch plantaris muscles. Sham and overloaded plantaris muscle AMPK phosphorylation (Thr172) was significantly higher in aged animals receiving normal chow compared to young adult animals; however, leucine supplementation in old animals reduced this AMPK phosphorylation to levels similar to young adult animals. Phospho-p70S6k (Thr389) and phospho-rpS6 (Ser235/Ser236) were significantly lower in old vs. young overloaded muscles under placebo conditions, but leucine partially restored both p70S6k and rpS6 phosphorylations in old overloaded muscles to that of young adult overloaded muscles. Overload significantly increased total eEF2 content and decreased inhibitory eEF2 phosphorylation (Thr56; normalized to total eEF2) in young adult muscles regardless of leucine supplementation. Total eEF2 content was unaffected by overload in old placebo muscles, but leucine supplementation in old animals non-significantly (p = 0.09) restored the overload-induced increase in total eEF2 content. Muscle eEF2 phosphorylation was unaffected by overload or leucine supplementation in old animals.

Discussion: These novel findings indicate that a leucine-enriched diet may potentially enhance overload-induced growth of aged fast-twitch muscle, in part by enhancing pathways known to stimulate protein synthesis. This is in accord with previous findings of leucine’s stimulating effect on protein synthesis in both young adult and aged skeletal muscle under resting conditions. The fact that leucine supplementation enhanced overload-induced hypertrophy only in the old (and not the young) animals may reflect the high growth stimulus of the chronic overload model. That is, the balance of protein synthesis/degradation rates under such a large chronic growth stimulus may not be the limiting factor in young animals, in which muscle growth is not impaired (i.e., synthesis/degradation rates may reach futile levels, and another factor such as sarcomere assembly may be limiting). However, the impaired balance of protein synthesis/degradation rates may be the limiting factor to growth in aged muscle, and leucine may correct this imbalance to restore muscle growth to levels observed in young animals.

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